Abstract

Objectives

To outline the development and validation of a universal method for quantifying deltamethrin, permethrin and alpha-cypermethrin levels in a variety of long-lasting insecticidal mosquito nets.

Methods

Using the HPLC conditions found in the CIPAC method for deltamethrin quantification, the method is based on a simple extraction technique for sample preparation (heating in isooctane at approximately 100 °C for 15 min). The method was validated for linearity, specificity, accuracy, precision, insecticide stability to extraction conditions and required extraction time for insecticide removal.

Results

The method was found valid for insecticide quantifications for various types of nets, namely for deltamethrin coated on polyester nets, deltamethrin incorporated into polyethylene nets, permethrin incorporated into polyethylene nets, alpha-cypermethrin coated on polyester nets and alpha-cypermethrin incorporated into polyethylene nets.

Conclusions

This method will provide a more simplified approach to testing a variety of nets (different types of fibre) containing deltamethrin, permethrin or alpha-cypermethrin.

Introduction

Long-lasting insecticidal mosquito nets (LN) continue to be a highly procured commodity for preventing the spread of malaria. An estimated 50 million nets are collectively procured annually by groups such as the United States Agency for International Development (USAID), Population Services International (PSI) and UNICEF. The World Health Organization's Pesticide Evaluation Scheme (WHOPES) has evaluated various types of nets for insecticide regeneration, wash resistance and field trials/real-life use (WHO, LN 2005, 2012a,b). Table 1 lists a variety of LN products available for use.

Pyrethroids, such as deltamethrin, permethrin and alpha-cypermethrin, are the predominant insecticides used (see Figure 1 for chemical structures). Deltamethrin comprises a single stereoisomer, namely (S)-α-cyano-3-phenoxybenzyl (1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate. The R-isomer [R, 1R, 3R] is a common impurity with lower biological activity (WHO, D 2009). Permethrin (3-phenoxybenzyl(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate) comprises two pairs of diastereoisomers (two cis and two trans). Permethrin used for LNs has a cis/trans ratio of 40:60 (WHO, P 2009). Cypermethrin has four pairs of diastereoisomers, with alpha-cypermethrin specifically being a racemic mixture of (S)-α-cyano-3-phenoxybenzyl (1R,3R)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate and (R)-α-cyano-3-phenoxybenzyl(1S,3S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (WHO, AC 2009a).

Laboratories that evaluate LNs may not have access to both types of chromatographic equipment. This work outlines the development and validation of a method for quantifying deltamethrin, permethrin and alpha-cypermethrin levels in LNs based on a simple extraction technique, where samples are analysed using the HPLC conditions found in the CIPAC method for deltamethrin quantification (CIPAC, D, Coated, 2009).

The average content of the insecticide should be within ± 25% of the target level (typically based on at least five samples).

Vestergaard Frandsen

1

100

White

Polyester

Deltamethrin

1.4

2

100

Blue

Polyester

Deltamethrin

1.4

3

75

White

Polyester

Deltamethrin

1.8

Bestnet

1

115

Light blue

Polyethylene

Deltamethrin

1.8

2

100

Blue

Polyethylene

Deltamethrin

1.8

3

115

Light blue

Polyethylene

Deltamethrin

1.8

4

118

Light blue

Polyethylene

Deltamethrin

1.8

Sumitomo

1

150

White

Polyethylene

Permethrin

20

2

150

Blue

Polyethylene

Permethrin

20

3

150

White

Polyethylene

Permethrin

20

4

150

White

Polyethylene

Permethrin

20

BASF

1

100

White

Polyester

Alpha-cypermethrin

5.0

Tana Netting

1

100

White

Polyester

Deltamethrin

2.0

Clarke

1

150

Green

Polyethylene

Alpha-cypermethrin

5.8

Test method summary

The method that was validated in this work is described below for a single sample of net. The approaches used for the validation are described in subsequent experimental sections. In practice, multiple samples of nets can be tested individually within a net and/or from multiple nets for a more statistically representative analysis. WHO specifications recommend to cut five pieces from a net (one on each side) and to combine them to form a representative sample of the net.

A 10 × 10 cm sample was cut from the net, followed by cutting into smaller pieces (about 16) and recording the total mass (necessary for calculating the mass of insecticide (g) per mass of net [kg]). The samples were transferred into a 100 ml boiling flask, followed by 50 ml of isooctane. All pieces of the net were submerged in the liquid. The flask was attached to a condenser and placed in a boiling water bath for 15 min for insecticide extraction. Afterwards, the flask was removed from the water bath and allowed to cool. The solution (without the net) was transferred into a 100 ml volumetric flask. With the net sample remaining in the boiling flask, the contents were rinsed twice with 20 ml isooctane, where these aliquots were added to the volumetric flask and the flask was diluted to 100 ml with isooctane. A portion of this solution was filtered through a 0.45 μm PTFE syringe filter into an HPLC vial. Based on the same sample preparation for all nets, the different types of nets have a range of target insecticide concentrations in the final sample solution (Vestergaard Frandsen, 0.0055 mg D/ml; Bestnet, 0.007 mg D/ml; Tana Netting, 0.008 mg D/ml; Sumitomo, 0.09 mg P/ml; BASF, 0.02 mg AC/ml; Clarke, 0.025 mg AC/ml). Standards for HPLC analysis were prepared from stock solutions of insecticide in isooctane (0.5 mg D/ml, 1.0 mg P/ml, 0.25 mg AC/ml) that were diluted to the desired target concentration for the type of net to be analysed. The HPLC system conditions incorporate a mobile phase of 95% isooctane and 5% 1,4-dioxane (with 0.15% water), a 1.3 ml/min flow rate, a Lichrosorb Si60, 5 μm, 150 × 4.6 mm analytical column at ambient temperature, a 20 μl injection volume (note – CIPAC method utilises a 5 μl injection) with a 10-min run time per injection and an analysis wavelength of 236 nm. Insecticide content (g/kg) of the net is calculated using the following formula: Content (g/kg) = (An/As) × Cs × (Vn/ms), where An is the area of the insecticide peak in net sample, As is average area of the insecticide peak in the working standards (from a single point calibration prepared at the target concentration), Cs is average concentration of the working standards (mg/ml), Vn is volume of sample solution (100 ml) and ms is mass of net sample (g). Permethrin levels are calculated by adding the areas for the cis- and trans-isomers, but the isomers may possibly be calculated separately if necessary.

Linearity/precision

Linearity was evaluated for each insecticide with duplicate sets of standard solutions that ranged from approximately 25 to 150% of the effective target concentration (deltamethrin, 0.002–0.010 mg/ml; permethrin, 0.02–0.13 mg/ml; alpha-cypermethrin, 0.005–0.030 mg/ml). From triplicate injections, the average peak area and percentage of relative standard deviation (%RSD) were determined for each standard solution. For each set of standards, a linear regression curve was obtained from the average areas, and the slope, intercept and r2 were determined. Precision was evaluated based on the %RSD of replicate injections and slope agreement between sets of standards.

Specificity

Various nets containing insecticide were extracted and analysed with HPLC (using the HP1200 with the diode array detector). Insecticide peaks from the samples were evaluated for the presence of obvious interferences (shoulders), and retention times were compared with that of insecticide standard injections. Insecticide peaks from the samples were analysed for peak purity (Stahl 2003), where the similarity factor for five spectra within the analyte peak (two on the peak front, one on the apex and two on the peak tail) was determined. Additionally, various net samples without insecticide (net blanks) were extracted and prepared for HPLC injection in the same manner as a normal sample. For each sample type, duplicate samples were prepared and each was injected three times.

Accuracy/precision

From the different types of blank nets, two samples (10 × 10 cm net) were prepared in the same manner (extracted and diluted to 100 ml) as a normal sample. To evaluate the accuracy for each insecticide, solutions with concentrations ranging from 50 to 150% were prepared by adding appropriate aliquots of stock standard into separate 10 ml volumetric flasks and filling to volume with the final solution obtained from the extracted blank net. Duplicate sets of sample solutions were prepared for each concentration, respectively, from the duplicate solutions obtained from the blank nets, where each sample solution was injected three times. Recoveries were evaluated by calculating the percentage of the measured insecticide level relative to the theoretical level from the addition of stock standard.

Insecticide stability to heat

For each insecticide type, 50 ml of solutions containing insecticide at various concentrations in isooctane (deltamethrin – 0.007, 0.014 and 0.021 mg/ml; permethrin – 0.09, 0.18 and 0.27 mg/ml; alpha-cypermethrin – 0.02, 0.04 and 0.06 mg/ml) was heated at approximately 100 °C for 15 min and subsequently diluted to 100 ml for HPLC analysis. Unheated insecticide standards (at the same respective concentrations of the heated standards subsequently diluted to 100 ml) were analysed by HPLC for area count comparison with heated insecticide solutions.

Effect of extraction time

From various types of nets, two samples of net were cut from adjacent positions on each respective net. The first sample was processed through a 15-min extraction for subsequent analysis with HPLC. The netting material was kept and processed through an additional 15-min extraction with fresh isooctane for HPLC analysis. The second sample was extracted for 30 min continuously, followed by HPLC analysis.

Results

Using the chromatographic conditions of the method, theoretical plates for all insecticides were approximately 8000, with tailing factors on the order of 1.2. Retention times for cis-permethrin, trans-permethrin, alpha-cypermethrin and deltamethrin were observed to be approximately 2.8, 3.1, 5.1 and 5.9 min, respectively. Resolution between cis- and trans-permethrin was observed to be approximately 2.5 and 1.9 for R-isomer and deltamethrin. For samples of alpha-cypermethrin nets (BASF and Clarke), there was a small peak observed in front of the alpha-cypermethrin peak that yielded a resolution of 1.7 relative to the main alpha-cypermethrin peak. Preliminary work with guard columns installed for column protection found that the resolution for deltamethrin and alpha-cypermethrin injections could be below 1.5, thus not providing adequate separation for the primary insecticide peak. Thus, guard columns have not been used for this work, but could possibly be if levels of impurities are negligible and additional column protection is desired. The effect of filtration was important to investigate to provide protection to the chromatographic system. PTFE syringe filters were found to be an adequate means of filtration because area counts (standards/samples) for filtered solutions were within 1% of area counts for unfiltered solutions, regardless of insecticide type.

Linearity/precision

Linearity was evaluated through the range of concentrations that encompass approximately 25–150% of the target sample concentration for each respective insecticide (Table 3), where a duplicate set of standards were prepared for each insecticide. With permethrin having both the cis- and trans-isomers, the linearity found for each respective isomer is provided with that found for the total permethrin. Coefficients of determination (r2) for all data sets indicate a high level of linearity, with Y-intercepts effectively proceeding through zero. For each insecticide, slope agreement was within 1% and (although not provided in Table 3) replicate injections for each standard solution were typically below 1% relative standard deviation. Figure 2 is a plot of the linearity data for each insecticide (deltamethrin, alpha-cypermethrin and total permethrin) and the approximate target concentration for the various net types.

Figure 2. Linearity data obtained for deltamethrin, permethrin and alpha-cypermethrin, with target insecticide concentrations indicated for various types of nets.

Specificity

Table 4 provides the results from sample injections of various nets containing insecticide, where different aspects were evaluated for specificity. No obvious interferences in sample peaks were observed, and sample retention times matched well with standards. Furthermore, peak purity analysis indicates that the insecticide peak from the samples evaluated has essentially no detectable contribution from other components. Additionally, various net samples without insecticide (net blanks) were extracted and prepared for HPLC injection in the same manner as a normal sample. Figures 3-5 show an example chromatogram from each of the blank net samples and example chromatograms for standards and net samples with insecticide (for reference). None of the sample injections showed any evidence of other analytes that would cause interference with the analysis of the respective insecticide. From this evaluation, there are no known interferences from any of the net materials evaluated thus far that cause any quantification issues for the insecticides.

No obvious interferences or shoulders with the insecticide peak in the sample injections.

b

Percentage retention time – 100% × Rt (smpl)/Rt (std).

c

Determined the average similarity factor for the 5 spectra within the analyte peak (2 on the peak front, 1 on the apex and 2 on the peak tail) where an ideal similarity factor for a pure analyte peak in the chromatogram is 1000 (Stahl 2003).

Deltamethrin

Vestergaard – White/100 (1)

None

99.6

>999.9

Vestergaard – Blue/100 (2)

None

99.9

>999.9

Bestnet – Light Blue/115 (1)

None

100.2

>999.7

Bestnet – Blue/100 (2)

None

100.6

>999.9

Permethrin (cis)

Sumitomo – White/150 (1)

None

99.9

>999.9

Sumitomo – Blue/150 (2)

None

99.9

>999.9

Sumitomo – White 150 (3)

None

100.0

>999.9

Permethrin (trans)

Sumitomo – White/150 (1)

None

99.9

>999.9

Sumitomo – Blue/150 (2)

None

99.8

>999.9

Sumitomo – White/150 (3)

None

100.1

>999.9

Alpha-Cypermethrin

BASF – White/100 (1)

None

100.1

>999.9

Accuracy/precision

Table 5 shows the insecticide recoveries for samples prepared by spiking extracts of blank nets with known levels of insecticide. Regardless of the insecticide or net type, all percentage of recoveries at each percentage of target concentration level were found within 98–102%, with overall %RSD generally being within 2%, thus indicating a high level of method accuracy and precision.

Table 5. Recoveries obtained from sample extracts of blank nets spiked with known levels of insecticides

Insecticide stability to heat

Pyrethroids are known to degrade or isomerise upon exposure to various environmental factors (Maguire 1990; Perschke & Hussain 1992; Liu et al. 2005). The effect of heat, as experienced during the sample preparation for this method, was evaluated to ensure that insecticide degradation (isomerisation) was not detectable. Solutions at 50, 100 and 150% of the target insecticide concentration during typical extraction (in 50 ml of isooctane) were prepared from insecticide standards (without nets), heated at approximately 100 °C for 15 min and diluted to 100 ml in isooctane for HPLC analysis. The percentage area counts of the heated solutions relative to unheated solutions (of the same respective concentration) were determined (Table 6). Area counts for heated standards were within 2% of those for the unheated standards. No evidence of insecticide degradation/isomerisation was observed, indicating that the heating experienced by the insecticide during extraction does not appear to alter the level of insecticide.

Effect of extraction time

The effect of extraction time was evaluated to determine the optimal extraction time for the removal of the insecticide during the sample preparation. Table 7 shows the results obtained for two sequential 15-min extractions on the same sample of net and for a 30-min extraction on an adjacent sample of net. The results indicate that the additional extraction of the same piece of net with fresh isooctane removes insecticide < 2% of the value obtained from the first 15-min extraction. Furthermore, a full 30-min extraction of sample provides very comparable values to those obtained for the adjacent sample extracted for only 15 min. Additionally, the remaining net samples (Lot 1 for Bestnet, Sumitomo and Clarke) that had been extracted for 30 continual minutes were analysed with the CIPAC method (CIPAC, D, Incorporated, 2009) that utilises the xylene extraction, which dissolves polyethylene. From this analysis, effectively no evidence was observed for deltamethrin and alpha-cypermethrin, and only approximately 0.3% of permethrin was found remaining. For all net types, the results indicate that a 15-min extraction with isooctane is adequate for complete insecticide removal.

Table 7. Effect of extraction time on the removal of insecticide from net materialsa

Although LNs are allowed to have an average insecticide level within 25% of the target value, the actual g/kg values (grams of insecticide/kg of net) of the nets used in the evaluation of extraction time are not reported to prevent comparison with the respective target values, thus protecting the confidentiality of all manufacturer's results. The purpose of this work is to develop and validate a method for insecticide quantification and is not to report the insecticide levels of actual nets, which may be used to judge the ability of a supplier to meet the specification. It should be noted that the values of g/kg found covered the expected range for the various insecticide/fibre types.

b

EX15A (g/kg) – First 15-min. extraction (Sample 1).

c

EX15B (g/kg) – Subsequent 15-min. extraction (Sample 1).

d

EX30 (g/kg) – 30-min. continuous extraction (Sample 2).

Vestergaard Frandsen

Blue/100 (2)

0.0

97.8

White/75 (3)

0.0

98.2

Tana Netting

White/100 (1)

0.0

103.9

White/100 (1)

0.0

100.6

Bestnet

Light Blue/115 (1)

0.0

96.3

Light Blue/115 (4)

0.0

101.0

Light Blue/115 (3)

0.0

97.8

Sumitomo

White/150 (1)

0.3

100.0

White/150 (4)

0.4

99.9

Blue/150 (2)

2.3

97.2

BASF

White/100 (1)

0.2

99.8

Clarke

Green/150 (1)

0.5

99.3

Discussion

The CIPAC methods employ a variety of extraction conditions depending on the insecticide/fibre combination. The intent of the extraction procedure for this method is to provide a more universal and simplified approach for sample preparation, regardless of the insecticide or fibre type. The CIPAC method for permethrin uses heptane exposure at 85–90 °C for 45 min for extraction from polyethylene (CIPAC, P). The CIPAC method (CIPAC, D, Incorporated, 2009) for quantifying deltamethrin in polyethylene refluxes the sample in xylene (at approximately 140 °C). The polyethylene dissolves in xylene at this elevated temperature, but precipitates upon cooling to room temperature, causing difficulty in sample handling. Moreover, xylene has to be changed to the mobile phase (isooctane/dioxane 95/5, v/v) before HPLC analysis. For this method, isooctane was chosen as the extraction solvent because it is the main component in the mobile phase for the HPLC analysis, minimising the number of reagents required. Additionally, extraction in isooctane at approximately 100 °C for 15 min provides a means of removing insecticide, from either polyester or polyethylene without dissolving fibre. Additionally, the CIPAC methods for deltamethrin quantification from both polyester and polyethylene utilise an internal standard (dipropyl/dibutyl phthalate) in the final sample solution without bringing the sample to volume in a volumetric flask. To simplify the number of reagents used for this method, an internal standard was not used, but rather the sample is brought to volume in a volumetric flask after extraction.

The method has been evaluated for linearity, specificity, accuracy, precision, insecticide stability to extraction conditions and required extraction time for insecticide removal. The method is considered appropriate for insecticide quantifications for various types of nets, namely for deltamethrin coated on polyester (Vestergaard Frandsen and Tana Netting), deltamethrin incorporated into polyethylene (Bestnet), permethrin incorporated into polyethylene nets (Sumitomo), alpha-cypermethrin coated on polyester (BASF) and alpha-cypermethrin incorporated into polyethylene (Clarke). The validity of using this analytical method for other nets, such as Vestergaard Frandsen nets comprised of deltamethrin/polyethylene and Bayer nets comprised of deltamethrin/polypropylene, will be the subject of future research.

Acknowledgements

A portion of this work was financially supported by the President's Malaria Initiative through the USAID | DELIVER PROJECT. The authors thank Stephen Smith from the CDC (Division of Parasitic Diseases and Malaria) for helpful suggestions regarding further validation of the extraction time.